Gastrointestinal health, immunity and performance by dietary intervention

ABSTRACT

This invention relates to the improvement of gastrointestinal health, immunity and performance by direct dietary intervention with a composition comprising a glucan and/or a fucan, and relates in particular to the transfer of associated health benefits to offspring via glucan and/or a fucan supplementation of the maternal diet. Accordingly the present invention provides a composition comprising at least one glucan, at least one fucan, or at least one glucan and at least one fucan for use in improving or maintaining the gastrointestinal health or function of a progeny of a maternal animal by administration to the maternal animal; and a method for improving or maintaining the gastrointestinal health or function of a progeny of a maternal animal, the method comprising administering a composition comprising at least one glucan, at least one fucan, or at least one glucan and at least one fucan to the maternal animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. patent application continued from U.S.Ser. No. 13/321,412 filed on Apr. 30, 2012, which is a U.S. NationalStage application under 35 U.S.C. 371 of International ApplicationNumber PCT/EP2010/003088, filed in English on May 21, 2010, which claimsthe benefit of patent application IE 2009/0398 filed in English on May21, 2009. The entirety of each application is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to the improvement of gastrointestinal health,immunity and performance by direct dietary intervention with laminarinand/or alpha-fucan and the transfer of associated health benefits tooffspring via laminarin and/or alpha-fucan supplementation of thematernal diet.

In particular, the invention has the purpose of improving thenutritional, immunological and microbiological status of suckling andweaned offspring by supplementation of the maternal diet with laminarinand/or alpha-fucans. In another aspect, this invention relates to theutilisation of laminarin and/or alpha-fucan-containing preparations orfeedstuffs to improve the immune status and immune response in pigs,poultry, sheep, horses, rabbits, fish, cats, dogs, humans and othermonogastric subjects. Other aspects relate to the use of such compoundsto increase performance in livestock as manifested by increased weightgain and feed conversion indices in weaned stock through maternaltransfer of beneficial compounds in utero or via colostrum and breastmilk during suckling, following supplementation of the maternal dietwith laminarin and/or alpha-fucan.

In another aspect, the invention relates to manipulating the sterileconditions of the intestine of neonates by reducing totalmicrobiological populations or by selectively encourage beneficialbacteria and inhibit growth of pathogens within the gastrointestinalsystem. Another aspect relates to the increasing the production ofstraight chain volatile fatty acids and reducing the production ofbranched chain volatile fatty acids within the gut by increasingfermentation from carbohydrate substrate and reducing fermentation fromprotein substrate. Yet another aspect relates to the synthesis of longchain polyunsaturated fatty acids including conjugated linoleic acid andomega-3 fatty acids by selectively stimulating Bifidobacteria in theintestinal tract.

In another aspect, the invention relates to the upregulation of mucinand/or trefoil factor (TFF) production in-vivo, thereby enhancingprotection and stability of the gastrointestinal mucosa against insult,infection or injury.

BACKGROUND OF THE INVENTION

Paediatric clinicians and veterinarians are well-informed on theimportance of achieving optimal nutrition during pregnancy to achievesuccessful physical, cognitive and neural development. Several trialshave demonstrated the effects associated with deficiencies or toxicitiesof nutrients and other compounds on foetal development and itssubsequent phenological characterisation. This has highlighted theimportance of prenatal and perinatal dietary interventions to achieveoptimal development during the critical stages and a healthy growth ratepost partum.

To compound these issues, the publication of the Swann report (1969)encouraged a more stringent control of antibiotic usage in animal feedsdue to the risks associated with antibiotic resistance, specifically theimposing threat on public health. This led to the EU prohibition ofgrowth promoting antibiotics in animal feeds in January 2006. Theprohibition of these growth promoters created a void in the market forintensive farming producers and also presented an opportunity forsourcing of a natural, safe alternative. The inclusion of laminarinand/or alpha-fucan in lactation diets of pigs, poultry, horses, sheep,rabbits, fish, humans and other monogastric subjects will have a majoreffect on the critical immunological and microbiological status at andimmediately following parturition and will therefore have a major effecton consequent welfare, development and growth rates.

Algal beta-glucans, called laminarin, consist of beta (1→3)-D glucosylsubunits with occasional (1→6) linked branches. Laminarin from Laminariadigitata occurs as two homologous series of molecules, a minor G seriescontaining 22-28 glucosyl residues and a more abundant M seriesconsisting of 20-30 glucosyl residues linked to a mannitol residue.Laminarin from many species of Laminaria (including Laminariahyperborea) is relatively insoluble and consists of predominantly beta(1→3) chains while laminarin from Laminaria digitata is soluble andconsists of small but significant levels of beta (1→6) linked branches.(Read et al, 1996).

Yeast beta glucans are found in long linear chains of up to 1300-1500glucose residues linked by beta (1→3) bonds with a minor incidence ofbeta (1→6) chains. Laminarin has much smaller chain lengths (average=24residues) with occasional beta (1→6) branches, depending on the species.Laminaria digitata has the beta (1→6) branching which make the glucansderived from them water soluble. Other Laminaria species, like Laminariahyperborea, do not have this branching which makes the linear chainsaggregate and makes the glucans derived from it, insoluble.

Natural polysaccharides built essentially of sulfated alpha-L-fucoseresidues are known as fucoidan (or alpha-fucans). These are present inbrown algae, some echinoderms and are the second most predominantpolysaccharide in brown seaweed, like Ascophyllum nodosum and species ofLaminaria. alpha-Fucans have been extensively studied due to theirdiverse biological activities, since they are potent anticoagulant,antitumor, and antiviral agents.

The present invention encompasses the use of alpha-fucans, in particularthe fucans present in sea plants, such as the sea cucumber body wall; inparticular the alpha-fucan present in the cell walls of marine algae,and the egg jelly coat of sea urchin eggs. Ideally the present inventionutilises fucoidan, the alpha-fucan present in macroalgae.

OBJECT OF THE INVENTION

It is an object of this invention to provide a novel method ofcontrolling microbiological, immunological and performance relatedattributes of livestock such as pig, poultry, horse as well as rabbits,fish, cats, dogs and human neonates through maternal transfermechanisms, by ensuring early delivery of beneficial compounds atcritical growth stages. Another object is to provide prenatal dietaryintervention with a laminarin and/or alpha-fucan containing preparationin the maternal diet for delivery through prenatal exchange in-utero orby postnatal transfer in colostrum or breastmilk. Another object is toprovide a dosing regimen for laminarin and/or alpha-fucan containingpreparations for controlling microbiological, immunological andperformance related attributes of livestock such as pigs, poultry,horses, as well as rabbits, cats, dogs, fish and human.

It is a further object of the invention that the composition willbeneficially affect the immune response by altering the expression ofpro- and anti-inflammatory cytokines, leukocytes population andexpression of immunoglobulins, mucins and trefoil factors.

Further objects of the invention include increasing the production ofvolatile straight chained fatty acids and reducing production ofbranched chain fatty acids (such as valeric, isovaleric and isobutyricacids) by altering the microbiological profile in favour of one thatpreferentially metabolises carbohydrates as fermentation substrate.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda composition comprising at least one glucan, at least one fucan, or atleast one glucan and at least one fucan for use in improving ormaintaining the gastrointestinal health or function of a progeny of amaternal animal by administration to the maternal animal.

According to a second aspect of the present invention, there is provideda method for improving or maintaining the gastrointestinal health orfunction of a progeny of a maternal animal, the method comprisingadministering a composition comprising at least one glucan, at least onefucan, or at least one glucan and at least one fucan to the maternalanimal.

The composition may comprise at least one glucan. When the compositioncomprises more than one glucan, each glucan may be the same glucan or adifferent glucan. Optionally or additionally, the composition maycomprise at least one fucan. When the composition comprises more thanone fucan, each fucan may be the same fucan or a different fucan.Optionally, the composition comprises at least on glucan, at least onefucan, or a mixture or combination thereof.

Optionally, the composition is administered to the maternal animalperinatally, prenatally, and/or postnatally. By “prenatally” is meantduring the period of time extending from initiation (fertilisation) toapproximately 50% of the total gestational term. Prenatal improvement ormaintenance of the gastrointestinal health or function of the progenycan occur during prenatal administration. By “perinatally” is meantduring the period of time extending from approximately 50% of the totalgestational term to the time of birth. Perinatal improvement ormaintenance of the gastrointestinal health or function of the progenycan occur during perinatal administration. By “postnatally” is meantduring the period of time extending from the time of birth, and isintended to extend to the period post-weaning (the period following thetime that the progeny ceases to ingest maternal colostrum or milk).Postnatal improvement or maintenance of the gastrointestinal health orfunction of the progeny can occur during postnatal administration.

By “progeny” is meant the offspring of a maternal animal, and isintended to include offspring developing in utero during the prenatalperiod, and offspring developing ex vivo during the postnatal period.

By “glucan” is meant a polysaccharide molecule comprising at least twosaccharide monomers, optionally D-glucose monomers, wherein each monomeris linked to an adjacent monomer by a glycosidic bond. Thepolysaccharide molecule may be linear or branched i.e. thepolysaccharide molecule can be a straight-chain polysaccharide or abranched chain polysaccharide. Optionally, the glucan is a branchedchain glucan. The glucan may be an alpha glucan or a beta glucan.Optionally, the glucan is a beta glucan. By “beta glucan” is meant aglucan comprising at least one beta glycosidic bond. A glycosidic bondis intended to mean a glycosidic bond, wherein a carbon atom of a firstmonomer forms a bond, optionally a single order bond, with a carbon atomon an adjacent monomer. A beta glycosidic bond is intended to mean aglycosidic bond, wherein a functional group, optionally a hydroxylgroup, attached to a carbon atom of a first monomer extends above theplane of the monomer (equatorially). Optionally, the C1 carbon atom of afirst monomer forms a bond, optionally a single order bond, with the C6carbon atom on an adjacent monomer. Further optionally, the glucancomprises a beta (1→6) glycosidic bond, optionally an oxygen-containingbeta (1→6) glycosidic bond. Optionally, at least one glucan is beta(1→3, 1→6) glucan. Still further optionally, the glucan is laminarin.

By “fucan” is meant a polysaccharide, optionally a sulphatedpolysaccharide, comprising at least two fucose saccharide monomers,wherein each monomer is linked to an adjacent monomer by a glycosidicbond. The polysaccharide molecule may be linear or branched. Optionally,the fucan is a branched fucan. The fucan may be an alpha fucan or a betafucan. Optionally, the fucan is an alpha fucan. By “alpha fucan” ismeant a fucan comprising at least one alpha glycosidic bond. Aglycosidic bond is intended to mean a glycosidic bond, wherein a carbonatom of a first monomer forms a bond, optionally a single order bond,with a carbon atom on an adjacent monomer. An alpha glycosidic bond isintended to mean a glycosidic bond, wherein a functional group,optionally a hydroxyl group, attached to a carbon atom of a firstmonomer extends below the plane of the monomer (axially).

Optionally, the C1 carbon atom of a first monomer forms a bond,optionally a single order bond, with either the C3 or C4 carbon atom onan adjacent monomer. Optionally, the fucan is fucoidan.

Optionally, the glucan and/or the fucan is isolated from a brown alga,optionally brown seaweed. Optionally, the brown alga is a brownmacroalga. Optionally, the brown macroalga, optionally brown seaweed, isselected from Phaeophyceae, optionally selected from PhaeophyceaeLaminariales and Phaeophyceae Fucales. Further optionally, the brownalga, optionally brown seaweed, is selected from Laminariaceae,Fucaceae, and Lessoniaceae. Optionally, the brown macroalga, optionallybrown seaweed, is selected from Ascophyllum species, optionallyAscophyllum nodosum and Laminaria species, optionally Laminariadigitata, Laminaria hyperborea, Laminaria saccharina, Laminaria japonicaor Sargassum species.

Alternatively, the glucan and/or the fucan is isolated from a red alga,optionally red seaweed. Optionally, the red alga is a red macroalga.Optionally, the red macroalga, optionally red seaweed, is selected fromFlorideophyceae, optionally selected from Florideophyceae Gigantinales,optionally selected from Gigartinaceae.

Optionally, the composition is administered daily to the maternalanimal.

Optionally, the composition is administered, optionally daily, to thematernal animal in an amount such that about 3-50 milligrams of glucanper kilogram of body weight is administered to the maternal animal.Further optionally, the composition is administered, optionally daily,to the maternal animal in an amount such that about 2-40 milligrams offucan per kilogram of body weight is administered to the maternalanimal.

Optionally, the composition is administered, optionally daily, to theanimal in an amount such that about 3-50 milligrams of glucan perkilogram of body weight is administered to the animal. Furtheroptionally, the composition is administered, optionally daily, to theanimal in an amount such that about 2-40 milligrams of fucan perkilogram of body weight is administered to the animal.

Optionally, the animal is a monogastric animal. Further optionally, theanimal is selected from pigs, poultry, horses, sheep, rabbits, fish,cats, dogs, and humans.

By “improving or maintaining the gastrointestinal health or function” ismeant improving the physiological function or histology of thegastrointestinal tract and/or the microbiological population of thegastrointestinal tract. Moreover, gastrointestinal health or functioncan be improved or maintained at the molecular level by improving theimmunological state of the host. The improvement or maintenance ofgastrointestinal health or function is intended to prevent orprophylactically treat disorders associated with poor gastrointestinalhealth or function, such as Crohn's disease, irritable bowel syndrome,and other such chronic conditions. Other disorders associated with poorgastrointestinal health are less serious and can include food-bornepathogens and certain bacteria and viruses that often result indiarrhoea, poor stool quality, low birth weight or weight gain, or othersymptoms of poor gastrointestinal health.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the concentration of immunoglobulin, optionallyImmunoglobulin G, in the colostrum or milk of the maternal animal.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the concentration of crude protein in thecolostrum or milk of the maternal animal.

Optionally, the gastrointestinal health or function is improved ormaintained by decreasing bacterial, optionally pathogenic bacterial,infection in the progeny. Further optionally, the bacterial, optionallypathogenic bacterial, infection is an Enterobacteriaceae infection,optionally selected from 20 Salmonella and Escherichia coli.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the expression of cytokines, optionallyselected from tumour necrosis factor alpha, interleukin-1 alpha,interleukin-6, and trefoil factor 3.

Optionally, the gastrointestinal health or function is improved ormaintained by decreasing the concentration of volatile branched-chainfatty acids, optionally selected from isobutyric acid, valeric acid, andisovaleric acid.

Optionally, the gastrointestinal health or function is improved ormaintained by altering the concentration or activity of phagocytes,optionally leukocytes, neutrophils, eosinophils, monocytes, orlymphocytes, further optionally leukocytes, eosinophils or lymphocytes.Further optionally, the concentration or activity of leukocytes isincreased and/or the concentration or activity of lymphocytes isdecreased and/or the concentration or activity of eosinophils isdecreased.

According to a further aspect of the present invention, there isprovided a composition comprising at least one glucan, at least onefucan, or at least one glucan and at least one fucan for use inimproving or maintaining the gastrointestinal health or function of ananimal by administration to the animal in an amount such that about 3-50milligrams of glucan per kilogram of body weight is administered,optionally daily, to the animal; or by administration to the animal inan amount such that about 2-40 milligrams of fucan per kilogram of bodyweight is administered, optionally daily, to the animal.

According to a still further aspect of the present invention, there isprovided a method for improving or maintaining the gastrointestinalhealth or function of an animal, the method comprising administering acomposition comprising at least one glucan, at least one fucan, or atleast one fucan and at least one fucan to the animal in an amount suchthat about 3-50 milligrams of glucan per kilogram of body weight isadministered, optionally daily, to the animal; or by administration tothe animal in an amount such that about 2-40 milligrams of fucan perkilogram of body weight is administered, optionally daily, to theanimal.

Optionally, the composition further comprises a sugar, optionally adisaccharide, optionally selected from lactose, sucrose, lactulose, andmaltose. Further optionally, the composition further comprises sugar,optionally a disaccharide, optionally selected from lactose, sucrose,lactulose, and maltose.

Optionally, the gastrointestinal health or function is improved ormaintained by decreasing bacterial infection, optionally Escherichiacoli infection.

Optionally, the gastrointestinal health or function is improved ormaintained and prevents or prophylactically treats diarrhoea.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the expression of cytokines, optionally in thepresence of antigen. Further optionally, the antigen is bacterialantigen, optionally bacterial lipopolysaccharide. Optionally, thecytokine is selected from interleukin-6 and interleukin-8.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the expression of mucins, optionally mucin-2and/or mucin-4.

Optionally, the gastrointestinal health or function is improved ormaintained by decreasing the concentration of circovirus or parvovirus,optionally porcine circovirus or porcine parvovirus. Further optionally,the porcine circovirus is type-2 porcine circovirus.

Optionally, the gastrointestinal health or function is improved ormaintained by increasing the concentration of straight-chain volatilefatty acids.

The inventors have developed a composition consisting of a formulationof laminarin and/or alpha-fucans that has altering effects on: (I) guthistology, (II) gut microbiology, (III) pro- and anti-inflammatorycytokine expression, (III) neonatal serum immunoglobulin levels, (IV)mucin production, (V) trefoil factor production, (VI) nutritional andimmunological composition of colostrum and breast milk and (VII)performance indices. In addition, there were clear detrimental effect onintestinal Enterobacteria populations which has associated benefits inreduced morbidity and mortality rates from reduced infection andinflammation.

Accordingly, the present invention provides use of a compositioncomprising beta-glucans and/or alpha-fucans in a method of improvingneonatal and weanling gastrointestinal health and immunity through pre-and postnatal supplementation of the maternal diet. In preferredembodiments, beta-glucans and alpha-fucans may be derived from more thanone source including seaweed and some echinoderms. The seaweed may befrom the group consisting of Laminariaceae, Fucacea, Gigartinaceae orLessoniaceae.

The invention also provides use of a composition comprising beta-glucansand/or alpha-fucans:

-   -   in a method of producing a maternal dietary supplement or        feedstuff, for reducing gastrointestinal bacterial populations        in neonates and weanlings;    -   in a method of producing a maternal dietary supplement or        feedstuff, for reducing morbidity and mortality rates in        neonates and weanlings;    -   in a method of producing a maternal dietary supplement or        feedstuff, for improving digestive histology by increasing the        villus height, reducing the crypt depth or increasing the        overall villus height:crypt depth ratio in neonates and        weanlings;    -   in a method of producing a maternal dietary supplement or        feedstuff, for improving performance in the progeny of livestock        such as pigs, poultry, horses, as well as rabbits, fish, cats,        dogs and humans including an increase in average daily gain, an        increase in average daily feed intake and an improvement in feed        efficiency;    -   in a method of improving gastrointestinal health by encouraging        beneficial microflora, reducing pathogenic microflora and        improving performance in neonates and weanlings, by        supplementing maternal diets    -   in a method of upregulating the production of mucins and trefoil        factors by epithelial cells as a means of enhanced physical        protection of the gastrointestinal epithelium.

In a further aspect the invention provides methods of achieving theabove-mentioned effects by feeding a composition comprising beta-glucansand/or alpha-fucans to humans, non-human animals or poultry.

In a still further aspect, the invention provides:

-   -   a dosing regimen for preventing bacterial or viral infection and        inflammation in livestock such as pigs, poultry, horses, sheep        as well as rabbits, fish, cats, dogs and humans by directly        supplementing the diet or by supplementing the maternal diet in        pre- and postnatal periods with a composition comprising        beta-glucans and alpha-fucans;    -   a dosing regimen for improving the nutritional quality and        increasing the immunoglobulin levels of colostrum and breast        milk by supplementing the maternal diet in the pre- and        postnatal periods with a composition comprising beta-glucans        and/or alpha-fucans;    -   a dosing regimen for increasing neonatal serum immunoglobulin        levels by in-utero transfer of beneficial immunostimulatory        compounds across the placental membrane by supplementing the        maternal diet in the pre- and postnatal periods with a        composition comprising beta glucans and/or alpha fucans;    -   a dosing regimen for increasing neonatal serum immunoglobulin        levels through an increased uptake in colostrum or breast milk        by supplementing the maternal diet in the pre- and postnatal        periods with a composition comprising beta glucans and/or alpha        fucans;    -   a dosing regimen for reducing Enterobacteria, including E. coli,        populations in the digestive tracts of neonatal pigs, poultry,        horses, as well as rabbits, fish, cats, dogs, humans and other        monogastric subjects by supplementing the maternal diet in the        pre- and postnatal periods with a composition comprising beta        glucans and/or alpha fucans;    -   a dosing regimen for alleviating functional intestinal disorders        associated with weaning by supplementing the maternal diet in        the pre- and postnatal periods with a composition comprising        beta glucans and/or alpha fucans;    -   a dosing regimen for encouraging a healthy intestinal        microbiological profile in neonates and weanlings by selectively        encouraging a dominant ratio of beneficial bacteria and        selectively inhibiting the growth of pathogenic bacteria in the        period of bacterial colonisation of intestine immediately after        birth by supplementing the maternal diet in the pre- and        postnatal periods with a composition comprising beta glucans        and/or alpha fucans.

The dosing regimen for administration of laminarin may be a daily dosageadministered at greater than 3 milligrams of laminarin per kilogram ofbody weight per day to a maximum of 50 milligrams per kilogram of bodyweight per day.

The dosing regimen for administration of alpha fucans may be a dailydosage administered of greater than 2 milligrams per kilogram of bodyweight per day to a maximum of 40 milligrams per kilogram of body weightper day.

The dosing regimen for administration of a combination of laminarin andalpha fucans may be a daily dosage of laminarin administered greaterthan 3 milligrams per kilogram of body weight per day to a maximum of 50milligrams per kilogram of body weight per day in combination with adaily dosage of alpha-fucans greater than 2 milligrams per kilogram ofbody weight per day to a maximum of 50 milligrams per kilogram of bodyweight per day.

The invention also provides use of a composition comprising beta-glucansand/or alpha-fucans in a method:

-   -   for increasing straight chain volatile fatty acid production        in-vivo;    -   for reducing branched chain volatile fatty acid production        in-vivo and their excretion;    -   for increasing long chain polyunsaturated fatty acids production        in-vivo;    -   for improving immune status and response in immune-challenged        livestock such as pigs, poultry, horses, as well as rabbits,        fish, humans and other monogastric subjects.    -   for improving the immune status by increased expression of pro-        and anti-inflammatory cytokines, mucins and trefoil factors.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described by the use ofnon-limiting examples, and reference made to the accompanying drawings,in which:

FIG. 1 illustrates PCV2-specific antibody titres of piglets fed a basaldiet;

FIG. 2 illustrates PCV2-specific antibody titres of piglets fed a basaldiet supplemented with LAM+FUC;

FIG. 3 illustrates PCV2-specific antibody titres of piglets fed a basaldiet supplemented with LAM+FUC and WPI;

FIG. 4 illustrates the percentage lymphocyte population for pigletsweaned onto different diets and subsequently challenged with PCV2 andPPV;

FIG. 5 illustrates the average percentage eosinophil population inpiglets fed different diets and subsequently challenged with PCV2 andPPV (Day 14 PI);

FIG. 6 illustrates the average terminal weight (Kg) of pigs weaned ontodifferent diets and then challenged with PCV2 and PPV;

FIG. 7 illustrates the average PCV2 DNA copy number detected in faecesof piglets weaned onto different diets and subsequently challenged withPCV2 and PPV; and

FIG. 8 illustrates PCV2 DNA copy number of pigs weaned onto differentdiets.

THE EXAMPLES

The examples given are results of investigative research on the effectsof laminarin and/or fucoidan supplementation on porcine subjects as amodel for all monogastrics including humans, animals and poultry. Theexamples shown include trials carried out using seaweed extractcontaining laminarin and fucoidan in combination (hereafter referred toas SWE) or each of the compounds individually as laminarin (hereafterreferred to as LAM) or fucoidan (hereafter referred to as FUC).

Example 1 Materials and Methods Animals and Treatment

40 pregnant sows were assigned to 1 of 4 dietary treatments (n=10sows/treatment): (T1) basal lactation; (T2) basal lactation+100 g/dayfish oil (F.O.); (T3) basal lactation+1.8 g/day SWE; and (T4) basallactation+100 g/day F.O.+1.8 g/day SWE from day 109 of gestation untilweaning at 26 days. SWE contained daily doses of laminarin (1 g) andfucoidan (0.8 g). For the test subjects, diets were top-dressed dailywith the experimental supplement. At birth, weight was recorded and 3piglets were selected to represent the mean birth weight of the litter.These were weighed weekly until weaning. At weaning, 120 mixed sex pigs(3 pigs per litter; average weight=8.05±0.46 Kg) were selected andoffered starter diets for 21 days. Feed and water were available adlibitum throughout the experiment. Pigs were individually weighed on theday of weaning (day 0) and thereafter at 7, 14 and 21 days post weaning.Feed intake recorded on a daily basis.

Sample Collection

30 ml of colostrum and milk was collected from sows on days 0 and 12post farrowing. Blood samples were collected from the jugular veins of 2piglets/litter on day 5 and 12 of lactation for immunoglobulin analysis.Crude protein was determined in accordance with the Association ofOfficial Analytical Chemists (AOAC, 1995).

Quantification of Immunoglobulins

Immunoglobulin assays were performed using specific pig ELISAquantification kits (Bethyl Laboratories, Inc., Montgomery, Tex., USA).Porcine assays were performed on sow colostrum and milk and piglet serumas described by Ilsley and Miller (2005).

Analysis of Selected Microbial Populations

Digesta samples were aseptically removed from the caecum and colon ofeach pig post-slaughter. Populations of Bifidobacterium, E. coli andLactobacillus spp. were selectively isolated and enumerated according toPierce et al. (2006).

Volatile Fatty Acid (VFA) Analysis

Samples of digesta from the caecum and colon were recovered for VFAanalysis by a gas chromatographic method following the procedures ofPierce et al. (2006).

Histological Analysis

Sections of duodenum, jejunum and ileum were aseptically removed,excised and fixed in 10% phosphate-buffered formalin. Cross sections at5 μm thickness of each intestinal segment were stained with haemotoxylinand eosin. Villus height and crypt depth were measured using a lightmicroscope fitted with an image analyser (Image Pro Plus; MediaCybernetics, Bethesda, Md., USA).

Phagocytosis Estimation of Blood Cells by Flow Cytometry

The PHAGOTEST® kit (Orpegen Pharma, Heidelberg, Germany), measuring theuptake of unopsonised, FITC-labelled E. coli, was used to measure thephagocytosing activity in whole blood cells. Samples were analyzed usinga Dako Cyan-ADP flow cytometer (Dako, Glostrup, Denmark).

Ileum and Colon Gene Expression—RNA Extraction and cDNA Synthesis

Tissue samples were collected from the ileum and colon, rinsed withice-cold PBS and immediately placed into tubes containing RNAlater®(Ambion Inc, Austin, Tex.). Total RNA was extracted using a Gene EluteMammalian Total RNA Miniprep Kit (Sigma-Aldrich) and quantified using aNanoDrop-ND1000 Spectrophotometer (Thermo Fisher Scientific Inc. MA,USA). Purity was assessed by determining the absorbance ratio at 260 and280 nm. Total RNA was reverse transcribed (RT) utilising a First StrandcDNA Synthesis Kit (Fermentas) using oligo dT primers.

Quantitative Real-Time PCR (qPCR)

Quantitative real-time PCR (qPCR) assays were performed on cDNA sampleson a 7900HT ABI Prism Sequence Detection System (PE Applied Biosystems,Foster City, Calif.) using SYBR Green PCR Master Mix (AppliedBiosystems). All primers used for RT-PCR (IL-la, IL-6, IL-10, TNF-α,MUC2, TFF3, GAPDH, B2M, ACTB, PPIA and YWHAZ) were designed using PrimerExpress™ software. Amplification was carried out in 10 μl SYBR PCRMastermix, 1 μl forward and reverse primer, 8 μl DPEC treated water and1 μl of template cDNA. Dissociation analyses of the PCR product wasperformed to confirm the specificity of the resulting PCR products.

Results Colostrum and Milk Composition

Colostral IgG levels were significantly higher in SWE supplemented sows(p<0.01). Supplementation increased protein concentration in sow's milkon day 12 (p<0.05).

TABLE 1 Effect of dietary treatment on total solid, crude protein, crudefat and immunoglobulin concentrations of sow colostrum and milk FO 0g/day 100 g/day p-value SWE - 1.8 g/day No Yes No Yes SEM SWE FO SWE xFO Colostrum Total solids % 24.78 25.20 25.69 24.89 1.422 0.897 0.8340.673 Crude protein % 13.27 13.90 14.72 14.31 1.145 0.925 0.423 0.656Fat % 6.27 6.10 5.93 5.04 0.737 0.478 0.352 0.632 IgG (mg/ml) 62.5270.11 64.01 69.56 2.557 0.010 0.844 0.681 IgA (mg/ml) 10.30 8.93 10.508.69 1.022 0.105 0.980 0.819 IgM (mg/ml) 4.08 4.91 3.86 4.11 0.444 0.2380.263 0.513 Sows milk Total solids % 20.11 20.22 19.48 19.77 0.486 0.6870.273 0.851 Crude protein % 5.17 5.42 5.17 5.36 0.109 0.050 0.837 0.761Fat % 9.02 8.80 8.51 8.36 0.546 0.731 0.388 0.940 IgG (mg/ml) 0.37 0.440.45 0.49 0.056 0.297 0.204 0.772 IgA (mg/ml) 3.59 3.72 4.03 3.83 0.3310.914 0.370 0.584 IgM (mg/ml) 1.56 1.56 2.10 1.56 0.419 0.519 0.5140.526

Suckling Piglet Immunoglobulins

Piglets suckling SWE supplemented sows had significantly higher serumIgG concentrations on day 5 (p<0.01) and day 12 of lactation (p<0.05)and enhanced serum IgA concentrations on day 5 of lactation (p>0.05).

TABLE 2 Serum immunoglobulin concentrations in piglets sucklingsupplemented sows. FO 0 g/day 100 g/day p-value SWE - 1.8 g/day No YesNo Yes SEM SWE FO SWE x FO Immunoglobulins (mg/ml) Day 5 IgG 17.63 23.0220.98 22.80 1.253 0.006 0.213 0.160 IgA 3.00 3.24 2.02 3.02 0.278 0.0330.036 0.174 IgM 1.52 1.61 1.41 1.39 0.222 0.881 0.468 0.805 Day 12 IgG9.91 12.47 10.07 11.62 0.880 0.025 0.689 0.570 IgA 0.35 0.27 0.41 0.390.090 0.598 0.324 0.776 IgM 0.53 0.61 0.54 0.63 0.051 0.098 0.789 0.929

Suckling Piglet Performance

Piglets suckling SWE supplemented sows had significantly lower averagedaily gains (p<0.05) during week 1 of lactation. There were nosignificant differences in daily gains between birth and weaning. Littersize, litter weight, piglet birth weight and weaning weight were notinfluenced by sow dietary treatments.

TABLE 3 Effect of maternal SWE supplementation on litter size, litterweight, piglet live weight and average daily gain (ADG) +FO 0 100p-value +SWE 1.8 g/day No Yes No Yes SEM SWE FO SWE x FO Litter size, n12.40 12.40 12.40 12.35 0.710 0.968 0.958 0.968 Litter weight (Kg) 14.5915.80 16.04 14.7 0.958 0.924 0.861 0.154 Birth weight (Kg) 1.25 1.281.28 1.27 0.061 0.828 0.921 0.656 Piglet BW (Kg) Day 7 3.37 2.92 3.202.91 0.161 0.016 0.546 0.612 Day 14 5.41 4.73 5.16 4.80 0.233 0.0210.697 0.462 Day 21 7.31 6.58 6.67 6.52 0.280 0.093 0.185 0.274 Day 268.73 7.85 7.85 7.76 0.340 0.127 0.131 0.209 ADG (Kg/day) Day 0 to 70.230 0.195 0.236 0.216 0.014 0.045 0.319 0.589 Day 8 to 15 0.282 0.2630.274 0.259 0.013 0.185 0.635 0.804 Day 15 to 21 0.286 0.277 0.237 0.2600.018 0.674 0.053 0.353 Day 21 to 26 0.264 0.254 0.240 0.266 0.022 0.6940.779 0.384 Day 0 to 26 0.277 0.256 0.250 0.254 0.012 0.501 0.236 0.294

Post-Weaning Piglet Performance

Piglets from SWE supplemented sows had significantly higher ADG from day7-14 (p<0.05) and day 0-21 (p=0.063) and feed intake (p<0.05) betweendays 7-14 post weaning.

TABLE 4 Effect of maternal dietary supplementation with SWE and FO fromday 109 of gestation until weaning (day 26) on post-weaning performance.SWE FO p-value Treatment No Yes SEM No Yes SEM SWE FO ADG (Kg/day) Day 0to 7 0.091 0.104 0.018 0.089 0.106 0.018 0.634 0.518 Day 7 to 14 0.2820.335 0.017 0.278 0.340 0.017 0.042 0.016 Day 14 to 21 0.450 0.476 0.0190.485 0.441 0.017 0.351 0.115 Day 0 to 21 0.275 0.308 0.012 0.284 0.2990.012 0.063 0.403 ADFI (Kg/day) Day 0 to 7 0.169 0.174 0.013 0.167 0.1750.013 0.781 0.691 Day 7 to 14 0.366 0.424 0.017 0.394 0.396 0.017 0.0250.932 Day 14 to 21 0.669 0.669 0.050 0.655 0.713 0.050 0.669 0.417 Day 0to 21 0.401 0.433 0.019 0.405 0.428 0.019 0.186 0.288 Gain:feed ratioDay 0 to 7 0.444 0.532 0.080 0.456 0.519 0.080 0.439 0583 Day 7 to 140.764 0.779 0.030 0.699 0.844 0.030 0.719 0.002 Day 14 to 21 0.692 0.7410.032 0.755 0.678 0.032 0.289 0.107 Day 0 to 21 0.634 0.692 0.030 0.6390.686 0.030 0.258 0.407

Microbiology

In the colon, maternal SWE supplementation resulted in a significantdecrease in Bifidobacteria populations (p<0.01). Furthermore, SWEsupplementation had a tendency to decrease E. coli and Lactobacilluspopulations in the colon compared to the control (p=0.09).

TABLE 5 Effect of maternal dietary supplementation with SWE and FO fromday 109 of gestation until weaning on selected intestinal microflora inthe 9 day old weaned pig. FO (g/day) 0 100 p-value SWE (1.8 g/day) NoYes No Yes SEM SWE FO SWE x FO Caecum (Log₁₀ CFU/g digesta)Bifidobacteria spp. 8.52 8.57 8.54 8.30 0.211 0.652 0.563 0.506Lactobacilli spp. 8.15 8.14 8.41 7.93 0.328 0.466 0.926 0.486 E. coli4.89 3.67 3.37 3.78 0.387 0.311 0.081 0.048 Colon (Log₁₀ CFU/g digesta)Bifidobacteria spp. 8.91 8.53 9.32 8.11 0.276 0.008 0.998 0.148Lactobacilli spp. 8.50 8.33 8.99 8.01 0.322 0.087 0.775 0.222 E. coli5.51 4.62 5.16 4.38 0.473 0.093 0.535 0.917

Cytokine Gene Expression

In the ileum of the post weaned pig, maternal SWE supplementationinduced a significant increase in the expression of the pro-inflammatorycytokine TNF-α (p<0.01). A significant increase in TFF 3 gene expressionwas also observed in the colon (p<0.05).

TABLE 6 Effect of maternal dietary supplementation with SWE from day 109of gestation until weaning on selected gene expression in the ileum andcolon of the weaned pig. SWE FO p-value Treatment No Yes SEM No Yes SEMSWE FO Ileum IL-1α 0.216 0.215 0.034 0.224 0.206 0.034 0.984 0.741 IL-60.212 0.166 0.032 0.197 0.181 0.032 0.325 0.747 TNF-α 0.164 0.575 0.1020.264 0.475 0.106 0.010 0.182 IL-10 0.127 0.075 0.023 0.085 0.116 0.0230.122 0.371 MUC 2 0.518 0.724 0.132 0.635 0.608 0.132 0.281 0.859 TFF 30.585 0.708 0.076 0.664 0.629 0.076 0.266 0.766 Colon IL-1α 0.150 0.1320.025 0.099 0.182 0.025 0.632 0.029 IL-6 0.170 0.124 0.026 0.102 0.1930.102 0.236 0.024 TNF-α 0.242 0.206 0.026 0.214 0.234 0.026 0.338 0.592IL-10 0.132 0.077 0.022 0.089 0.121 0.022 0.092 0.324 MUC 2 0.490 0.5080.095 0.616 0.381 0.095 0.733 0.182 TFF 3 0.371 0.565 0.068 0.536 0.4000.068 0.045 0.111Volatile Fatty Acid (VFA) Analysis and pH measurement

TABLE 7 Effect of maternal dietary treatment with SWE and FO from day109 of gestation until weaning on VFA composition of intestinal contentsof the 9 day old weaned pig. FO (g/day) 0 100 p-value SWE (1.8 g/day) NoYes No Yes SEM SWE FO SWE x FO Caecum (mmol/g digesta) Total VFA 181.7168.0 170.4 183.2 11.20 0.968 0.865 0.249 Acetic acid 0.660 0.645 0.6750.665 0.011 0.289 0.124 0.801 Propionic acid 0.228 0.245 0.240 0.2450.010 0.298 0.539 0.542 Butyric acid 0.093 0.089 0.063 0.074 0.008 0.6640.009 0.371 Isobutyric acid 0.003 0.003 0.004 0.002 0.001 0.308 0.9490.295 Valeric acid 0.011 0.012 0.012 0.010 0.002 0.823 0.686 0.492Isovaleric acid 0.005 0.006 0.006 0.004 0.001 0.582 0.572 0.264Acetic:propionic acid 2.94 2.68 2.84 2.75 0.158 0.287 0.897 0.624 BCFAs*0.020 0.021 0.022 0.016 0.003 0.482 0.623 0.226 pH 6.17 6.27 6.41 6.070.189 0.511 0.922 0.255 Colon (mmol/g digesta) Total VFA 151.6 146.0128.1 168.7 12.62 0.177 0.974 0.080 Acetic acid 0.658 0.631 0.672 0.6630.014 0.209 0.113 0.521 Propionic acid 0.216 0.229 0.281 0.232 0.3560.617 0.344 0.386 Butyric acid 0.087 0.010 0.087 0.077 0.012 0.799 0.2680.269 Isobutyric acid 0.007 0.008 0.012 0.006 0.002 0.080 0.317 0.043Valeric acid 0.011 0.016 0.019 0.012 0.002 0.705 0.471 0.038 Isovalericacid 0.012 0.013 0.021 0.010 0.002 0.053 0.222 0.028 Acetic:propionicacid 3.07 2.79 3.18 2.95 0.184 0.166 0.462 0.883 BCFAs* 0.030 0.0360.052 0.028 0.005 0.127 0.195 0.009 pH 6.28 6.17 6.48 6.44 0.119 0.5540.063 0.783 *BCFAs, branched chain fatty acids

Histology

In the ileum, there was a significant effect of SWE supplementation onvillus height and villus height to crypt depth ratio (p<0.05). Resultsfrom the duodenum also showed a beneficial effect emulating from SWEsupplementation on crypt depth (p>0.10)

TABLE 8 Effect of maternal dietary supplementation with SWE and fish oil(FO) from day 109 of gestation until weaning (day 26) on villus height,crypt depth and villus height to crypt depth ratio in the 9 day oldweaned pig. Fish oil (g/d) 0 100 p-value SWE (1.8 g/d) No Yes No Yes SEMSWE FO SWE x FO Villous height (μm) Duodenum 419.4 415.9 430.1 421.55.62 0.291 0.183 0.645 Jejunum 384.2 396.2 395.4 382.8 5.00 0.952 0.8430.022 Ileum 215.0 233.0 238.7 232.6 6.00 0.328 0.063 0.055 Crypt depth(μm) Duodenum 328.8 314.3 316.0 315.4 4.40 0.097 0.216 0.122 Jejunum288.6 280.3 291.7 288.1 6.87 0.392 0.458 0.731 Ileum 178.0 172.4 167.9171.7 4.75 0.853 0.270 0.333 Villous:crypt depth ratio Duodenum 1.281.31 1.36 1.32 0.02 0.788 0.049 0.164 Jejunum 1.33 1.43 1.36 1.33 0.030.288 0.177 0.034 Ileum 1.21 1.36 1.42 1.35 0.04 0.444 0.015 0.013

Phagocytosing Capacity

SWE supplementation exerted a suppressive effect on total eosinophilnumbers (p<0.01) in suckling piglets. Dietary SWE supplementationresulted in a higher percentage of E. coli phagocytosing leukocytes(p<0.05) and a lower percentage of E. coli phagocytosing lymphocytes(p<0.01) compared to non SWE-supplemented diets.

TABLE 9 Effect of dietary treatment on the phagocytosing activity (totalnumber and % positive phagocytosis) of piglet whole blood cells atweaning SWE FO p-value No Yes SEM No Yes SEM SWE FO Leukocytes 2247520912 6637 20896 22492 1637 0.595 0.365 Positive % 57.6 64 2.2 57.1 64.52.2 0.046 0.024 Lymphocytes 6650 5127 672 6292 5485 672 0.116 0.575Positive % 13.3 10.1 0.834 10.5 12.9 0.834 0.008 0.050 Monocytes 26412578 83 2575 2644 83 0.627 0.614 Positive % 74.1 77.7 2.8 72.7 79.1 2.80.369 0.112 Neutrophils 8650 9489 883 7977 10161 883 0.407 0.076Positive % 91.1 92.1 1.2 91.4 91.9 1.2 0.564 0.796 Eosinophils 512 33861 384 466 61 0.002 0.297 Positive % 26 21.8 2.1 23.2 24.6 2.1 0.1630.653

Example 2 Experiment 1 Materials and Methods Experimental Design andDiets

Experiment 1 was designed as a complete randomised design comprising offive dietary treatments as follows: (T1) 0 g/Kg SWE (control), (T2) 0.7g/Kg SWE, (T3) 1.4 g/Kg SWE extract, (T4) 2.8 g/Kg SWE extract and (T5)5.6 g/Kg SWE extract. The SWE contained LAM+FUC. All diets wereformulated to have identical concentrations of net energy and totallysine. The amino acid requirements were met relative to lysine (Close,1994). Chromic oxide was added at the time of milling to all diets atthe rate of 150 ppm for the determination of ash digestibility.

Animals and Management

30 finishing boars with an initial live weight of 51±3.4 Kg were used inthe experiment. The pigs were blocked on the basis of live weight andrandomly allocated to one of five dietary treatments. The pigs wereallowed a 14-day dietary adaptation period after which time they wereweighed and transferred to individual metabolism crates. Animals wereallowed a 5-day acclimatisation period, followed by a 5-day collectionperiod to facilitate an apparent digestibility and nitrogen balancestudy. The daily feed allowance (DE intake=3.44×(live weight)^(0.54)(Close, 1994) was divided over two meals. Water was provided with mealsin a 1:1 ratio. Between meals, fresh water was provided ad libitum. Themetabolism crates were located in an environmentally controlled room,maintained at a constant temperature of 22° C. (±1.5° C.).

Coefficient of Total Tract Apparent Digestibility (CTTAD) and NitrogenBalance Study

During collections, urine was collected in a plastic container, via afunnel below the crate, containing 20 ml of sulphuric acid (25% H₂S0₄).To avoid nitrogen volatilisation, the funnel was sprayed four timesdaily with weak sulphuric acid (2% H₂S0₄) solution. The urine volume wasrecorded daily and a 50 ml sample was collected and frozen forlaboratory analysis. Total faeces weight was recorded daily and ovendried at 100° C. A sample of freshly voided faeces was collected dailyand frozen for nitrogen analysis and pH measurement. At the end of thecollection period, the faeces samples were pooled and a sub-sampleretained for laboratory analysis. Feed samples were collected each dayand retained for chemical analysis. All 30 pigs remained on theirrespective dietary treatments until slaughter.

Example 2 Experiment 2 Materials and Methods Experimental Design andDiets

This experiment was designed as a 2×2 factorial design comprising fourdietary treatments: (T1) control diet, (T2) control+300 ppm LAM, (T3)control+238 ppm FUC, (T4) control+300 ppm LAM+238 ppm FUC. All dietswere standardised for net energy (9.8 MJ/Kg) and total lysine (10 g/Kg).Amino acid requirements were met relative to lysine (Close, 1994).

TABLE 10 Composition and analysis of diets - experiment 1 (as fedbasis). Treatment 1 2 3 4 5 Ingredients (g · kg⁻¹) Laminaria hyperboreaextract 0 0.7 1.4 2.8 5.6 Wheat 704.3 703.6 702.9 701.5 698.7 SoybeanMeal 265 265 265 265 265 Soya Oil 5.7 5.7 5.7 5.7 5.7 Mineral andVitamin^(†) 2.5 2.5 2.5 2.5 2.5 Limestone 15 15 15 15 15 Dicalciumphosphate 7.5 7.5 7.5 7.5 7.5 Analysed Composition (g · kg⁻¹) Laminarin0 0.075 0.150 0.300 0.600 Fucoidan 0 0.059 0.119 0.238 0.476 Dry Matter869.1 871.8 886.5 884.4 878.2 Crude Protein (N x 6.25) 215.7 203.2 199.5200.8 195.1 Neutral Detergent Fibre 119.7 97.8 91.8 96.4 98 Aciddetergent fibre 39.0 35.2 30.7 32.7 35.7 Crude Ash 45.7 48.4 47.7 51.252.6 Lysine 9.9 9.9 9.9 9.9 9.9 Methionine and cysteine^($) 6.0 6.0 6.06.0 6.0 Threonine 7.0 7.0 7.0 7.0 7.0 Tryptophan 1.9 1.9 1.9 1.9 1.9Calculated composition (g · kg⁻¹) Digestible Energy^(#) 13.9 13.9 13.913.9 13.8 Calcium 7.29 7.29 7.30 7.32 7.35 Phosphorus 4.04 4.04 4.044.04 4.04 ^(†)Provided per kg of complete diet: 3 mg retinol, 0.05 mgcholecalciferol, 40 mg alpha- tocopherol, 90 mg copper as copper IIsulphate, 100 mg iron as iron II sulphate, 100 mg zinc as zinc oxide,0.3 mg selenium as sodium selenite, 25 mg manganese as manganous oxideand 0.2 mg iodine as calcium iodate on a calcium sulphate/calciumcarbonate carrier.

Animals and Management

28 finishing boars with an initial live weight of 55 Kg were used. Pigswere blocked on the basis of live weight and were randomly allocated toone of four dietary treatments. The pigs were allowed a 28-day dietaryadaptation period after which time they were weighed and slaughtered.

Microbiology and Apparent Digestibility of Ash in the Proximate Caecumand Colon

Digesta was aseptically removed from the proximal caecum and colon ofeach animal after slaughter. Chromic oxide was used as marker todetermine ash digestibility in the caecum and colon. Bifidobacteriaspp., Lactobacillus spp. and Enterobacteria were isolated and countedaccording to the method described by O'Connell et al., (2005).

Volatile Fatty Acid Sampling and Analysis

Samples of digesta from the caecum and the proximal and distal colon ofindividual pigs were taken for VFA analysis. VFA concentrations in thedigesta were determined using a modified method of Porter and Murray(2001) according to O'Connell et al. (2005).

Results Experiment 1—Microbiology Study

TABLE 11 Effect of SWE concentration on microbial ecology and pH in thecaecum and colon Treatment 1 2 3 4 5 L. hyperborea extract (g/Kg) 0 0.71.4 2.8 5.6 s.e.m. Linear Quadratic Proximal Caecum BacterialPopulations (CFU/ml digesta) Enterobacteria spp. 6.94 7.15 6.65 6.426.70 0.196 ns * Bifidobacteria spp. 8.33 8.45 8.41 8.25 7.86 0.174 ** nsLactobacilli spp. 8.67 8.85 8.73 8.84 8.62 0.140 ns ns Proximal ColonBacterial Populations (CFU/ml digesta) Enterobacteria 6.95 6.72 6.346.49 6.85 0.245 ns * Bifidobacteria spp. 8.37 8.62 8.77 8.57 8.16 0.118ns ** Lactobacilli spp. 9.10 9.15 9.07 8.90 8.83 0.113 * ns Caecum pH5.63 5.90 6.42 5.49 5.69 0.125 ns *** Colon pH 5.94 6.11 6.18 5.85 5.940.079 ns ** * = (p < 0.05), ** = (p < 0.01), *** = (p < 0.001), ns = nonsignificant (p > 0.05), $ = (p < 0.1)

Experiment 1—Apparent Ash Digestibility

TABLE 12 Effect of SWE concentration on apparent nutrient digestibilityand nitrogen balance. Treatment 1 2 3 4 5 L. hyperborea SWE 0 0.7 1.42.8 5.6 s.e.m. Linear Quadratic Average Daily Feed 2158 2168 2138 22142209 * Intake (g/d) Laminarin Intake (mg/d) 0 162.6 320.7 664.2 1325.4Fucoidan Intake (mg/d) 0 127.9 254.4 526.9 1051.4 Water Intake (Kg/d)5.11 4.65 5.44 5.80 6.08 0.043 * ns Urine Output (Kg/day) 2.803 3.2563.654 3.445 4.273 0.320 * ns Nitrogen Intake (g/day) 64.72 61.48 60.4862.90 60.51 0.691 * ns Digestibility Coefficients Neutral DetergentFibre 0.66 0.55 0.56 0.58 0.56 0.012 ns * Nitrogen 0.90 0.90 0.89 0.900.89 0.006 ns ns Dry Matter 0.89 0.89 0.89 0.89 0.89 0.002 ns ns OrganicMatter 0.91 0.91 0.91 0.91 0.91 0.003 ns ns Ash DigestibilityCoefficients Caecal Ash 0.49 0.45 0.40 0.35 0.43 0.037 ns ** Colonic Ash0.56 0.49 0.51 0.50 0.51 0.016 ns ** Total Tract Ash 0.57 0.62 0.56 0.620.62 0.010 ** ns Nitrogen (N) Balance Faecal N Excretion 6.93 6.93 7.027.34 7.16 0.417 ns ns (g/day) Urinary N Excretion 29.18 28.91 29.6128.79 34.27 1.07  ns * (g/day) Total N Excretion (g/day) 36.14 35.8936.84 35.69 41.60 1.12  ns * N Retention (g/day) 25.87 26.12 25.18 26.3120.41 1.17  ns * * = (p < 0.05), ** = (p < 0.01), *** = (p < 0.001), ns= non significant (p > 0.05)

Experiment 1—Volatile Fatty Acid Study

TABLE 13 Effect of SWE concentration on concentration & molarproportions of VFAs Treatment 1 2 3 4 5 L. hyperborea SWE 0 0.7 1.4 2.85.6 s.e.m. Linear Quadratic Proximal Caecum (mmol/L) VFAs 281.2 241.7378.1 197.9 227.8 13.91 * ** Acetic acid 0.626 0.656 0.653 0.621 0.6290.011 ns ** Propionic acid 0.210 0.197 0.172 0.202 0.195 0.008 ns **Isobutyric acid 0.014 0.011 0.017 0.014 0.005 0.002 * * Butyric acid0.107 0.101 0.111 0.105 0.118 0.005 ns ns Isovaleric acid 0.020 0.0170.023 0.027 0.014 0.002 ns ** Valeric acid 0.019 0.016 0.021 0.019 0.0140.002 ns ns Acetic:Propionic 2.86 3.22 3.83 3.16 3.35 0.155 ns *** BCFAs0.052 0.044 0.062 0.060 0.034 0.006 ns * Proximal Colon (mmol/L) VFAs342.3 376.8 371.7 281.0 369.0 45.31 ns ns Acetic acid 0.579 0.575 0.5690.574 0.579 0.013 ns ns Propionic acid 0.201 0.196 0.200 0.206 0.1950.004 ns ns Isobutyric acid 0.023 0.027 0.025 0.025 0.026 0.005 ns nsButyric acid 0.123 0.132 0.133 0.130 0.126 0.006 ns ns Isovaleric acid0.034 0.038 0.038 0.034 0.039 0.003 ns ns Valeric acid 0.030 0.035 0.0330.028 0.033 0.005 ns ns Acetic:Propionic 2.801 3.032 2.839 2.784 2.8690.099 ns ns BCFAs 0.088 0.101 0.097 0.088 0.099 0.016 ns ns * = (p <0.05), ** = (p < 0.01), *** = (p < 0.001), ns = non significant (p >0.05)

Experiment 2—Microbiology Study

TABLE 14 Effect of LAM and FUC on the concentration and molarproportions of VFAs Treatment 1 2 3 4 Significance Control LAM FUCLAM/FUC s.e.m. LAM FUC LAM X FUC Proximal Colon (mmol/L) Total VFA 168.7173.6 195.5 197.9 7.925 ns ** ns Acetic acid 0.618 0.576 0.599 0.6470.011 ns * *** Propionic acid 0.213 0.268 0.251 0.217 0.011 ns ns ***Isobutyric acid 0.009 0.003 0.004 0.003 0.001 ** * * Butyric acid 0.1240.125 0.118 0.114 0.004 ns ns ns Isovaleric acid 0.017 0.011 0.011 0.0080.002 * * ns Valeric acid 0.017 0.017 0.014 0.011 0.002 ns ** nsAcetic:propionic 2.94 2.17 2.44 3.02 0.159 ns ns *** BCFAs 0.042 0.0310.026 0.022 0.004 * ** ns Distal Colon (mmol/L) Total VFA 126.02 136.7172.6 159.5 9.34 ns *** ns Acetic acid 0.597 0.571 0.599 0.636 0.012 ns** ** Propionic acid 0.195 0.203 0.186 0.181 0.005 ns *** ns Isobutyricacid 0.026 0.022 0.021 0.020 0.001 ns ** ns Butyric acid 0.118 0.1340.139 0.113 0.007 ns ns ** Isovaleric acid 0.040 0.034 0.034 0.0320.002 * * ns Valeric acid 0.024 0.023 0.021 0.018 0.001 ns *** nsAcetic:propionic 3.08 2.76 3.23 3.52 0.128 ns *** * BCFAs 0.089 0.0780.076 0.070 0.003 * ** ns

Experiment 2—Volatile Fatty Acid Study

TABLE 15 Effect of LAM and FUC concentration on concentration and molarproportions of VFAs Treatment 1 2 3 4 Significance Control LAM FUCLAM/FUC s.e.m. LAM FUC LAM x FUC Proximal Colon (mmol/L) Total VFA 168.7173.6 195.5 197.9 7.925 ns ** ns Acetic acid 0.618 0.576 0.599 0.6470.011 ns * *** Propionic acid 0.213 0.268 0.251 0.217 0.011 ns ns ***Isobutyric acid 0.009 0.003 0.004 0.003 0.001 ** * * Butyric acid 0.1240.125 0.118 0.114 0.004 ns ns ns Isovaleric acid 0.017 0.011 0.011 0.0080.002 * * ns Valeric acid 0.017 0.017 0.014 0.011 0.002 ns ** nsAcetic:Propionic 2.94 2.17 2.44 3.02 0.159 ns ns *** BCFAs 0.042 0.0310.026 0.022 0.004 * ** ns Distal Colon (mmol/L) Total VFA 126.02 136.7172.6 159.5 9.34 ns *** ns Acetic acid 0.597 0.571 0.599 0.636 0.012 ns** ** Propionic acid 0.195 0.203 0.186 0.181 0.005 ns *** ns Isobutyricacid 0.026 0.022 0.021 0.020 0.001 ns ** ns Butyric acid 0.118 0.1340.139 0.113 0.007 ns ns ** Isovaleric acid 0.040 0.034 0.034 0.0320.002 * * ns Valeric acid 0.024 0.023 0.021 0.018 0.001 ns *** nsAcetic:Propionic 3.08 2.76 3.23 3.52 0.128 ns *** * ratio BCFAs 0.0890.078 0.076 0.070 0.003 * ** ns * = (p < 0.05), ** = (p < 0.01), *** =(p < 0.001), ns = non significant (p > 0.05)

Example 3 Experimental Design and Diets

This experiment was carried out over two consecutive periods of 25 days.240 piglets were selected after weaning at 24 days and assigned to oneof four dietary treatments. Pigs in period 1 and 2 had initial liveweights of 7.2 Kg and 7.8 Kg (±0.9 Kg), respectively. This experimentwas designed as a 2×2 factorial. During the experiment (days 0-25)piglets were offered the following diets: (T1) 150 g/Kg lactose; (T2)150 g/Kg lactose+SWE; (T3) 250 g/Kg lactose (T4) 250 g/Kg lactose+SWE.SWE was included at 2.8 g/Kg and derived from Laminaria digitata. Itcontained laminarin (112 g/Kg), fucoidan (89 g/Kg) and ash (799 g/Kg).

Animals and Management

Pigs were housed in groups of 4 (n=15/treatment) and weighed at weaning(day 0), day 7, 14 and 25. Pigs were fed ad libitum. Fresh faecalsamples were collected on days 10 to 15 for determination of nutrientdigestibility and VFA analysis. Fresh faecal samples were collected onday 10 for enumeration of E. coli and Lactobacilli (O'Connell et al.,2005).

Microbiology

1 g of faecal sample was serially diluted in maximum recovery diluent(MRD; Oxoid, Basingstoke, UK) and plated on selective agars.Lactobacillus spp. were isolated on de Man Rogosa Sharp agar (MRS,Oxoid). The API 50 CHL (BioMerieux, France) kit was used to confirmsuspect Lactobacilli spp. E. coli species were isolated on MacConkeyagar (Oxoid). Suspect colonies were confirmed with API 20E (BioMerieux,France).

Results Performance

TABLE 16 The effect of lactose and SWE on piglet performance TreatmentSignificance 1 2 3 4 SEM Lactose SWE Lactose x SWE Lactose (g/Kg) 150250 SWE − + − + Average Daily Gain (ADG) (Kg/day) Day 0-7 0.100 0.1480.146 0.183 0.018 * * ns Day 7-14 0.302 0.303 0.325 0.387 0.021 * ns nsDay 14-25 0.438 0.427 0.388 0.455 0.020 ns ns * Day 0-25 0.275 0.2930.287 0.350 0.013 * ** ns Average Daily Feed Intake (ADFI) (Kg/day) Day0-7 0.242 0.257 0.239 0.271 0.015 ns ns ns Day 7-14 0.415 0.426 0.4500.472 0.019 * ns ns Day 14-25 0.682 0.663 0.683 0.737 0.025 ns ns ns Day0-25 0.446 0.449 0.458 0.502 0.014 * ns ns Gain: Feed ratio (Kg/Kg) Day0-7 0.413 0.558 0.589 0.659 0.062 * ns ns Day 7-14 0.747 0.705 0.7290.832 0.055 ns ns ns Day 14-25 0.633 0.636 0.569 0.622 0.027 * ns ns Day0-25 0.603 0.633 0.619 0.691 0.062 ns * ns Probability ofsignificance; * P < 0.05; ** P < 0.01, ns P > 0.05

Coefficient of Total Tract Apparent Digestibility (CTTAD)

TABLE 17 The effect of dietary treatment on the coefficient of totaltract apparent digestibility Treatment Significance 1 2 3 4 SEM LactoseSWE Lactose x SWE Lactose 150 250 (g/Kg) SWE − + − + Digestibility (%)DM 87.75 91.65 91.34 95.25 0.500 *** *** ns OM 89.16 92.52 92.24 95.820.535 *** *** ns N 83.69 89.59 86.86 92.34 1.038 * *** ns Ash 53.3070.60 72.80 83.08 2.140 *** *** ns GE 85.93 90.93 90.22 94.46 0.698 ****** ns NDF 37.55 65.01 61.91 74.60 2.970 *** *** * Probability ofsignificance; * p < 0.05; ** p < 0.01, *** p < 0.001, ns p > 0.05

Microbiology and VFAs

TABLE 18 Effect of dietary treatment on Lactobacilli and Escherichiacoli populations Treatment Significance 1 2 3 4 SEM Lactose SWE Lactosex SWE Lactose (g/Kg) 150 250 SWE − + − + Bacterial populations (Log₁₀CFU/g faeces) Lactobacilli 8.46 8.63 8.19 8.84 0.12 ns *** * Escherichiacoli 6.30 5.80 5.70 4.50 0.42 * * ns Probability of significance; * P <0.05; ** P < 0.01, ns P > 0.05

Example 4 Experimental Design and Diets

One hundred and ninety two piglets were weaned at twenty four days ofage, with an initial live weight of 6.4±0.785 Kg and assigned to one offour dietary treatments or 21 days post weaning. The dietary treatmentsconsisted of (T1) basal diet, (T2) basal diet with 300 ppm LAM, (T3)basal diet with 236 ppm FUC, (T4) Basal diet with 300 ppm LAM and 236ppm FUC. Diets were formulated to have identical concentrations ofdigestible energy (DE) (16 MJ/Kg) and ileal digestible lysine (14 g/Kg).All amino acid requirements were met relative to lysine (Close, 1994).Chromium III oxide was added to the diets for determination of nutrientdigestibility. The LAM and FUC were derived from Laminaria hyperborea.

Management

Piglets were housed in groups of 4 and offered feed twice daily. Waterwas supplied ad-libitum. Any pig displaying symptoms of illness wastreated appropriately and recorded. Pigs were weighed on day 0 (day ofweaning), 7, 14 and 21. Feed intake was monitored weekly. Fresh faecalsamples were taken on day 10 and were analysed for E. coli andLactobacilli concentrations. Faeces samples were collected from each penon day 12-17 and were retained for chemical analysis. Fresh faecalsamples were removed on day 14 and were frozen and retained for volatilefatty acid analysis. Fresh faecal samples were taken on day 17 for pHdetermination.

Faeces Scoring and Morbidity

Pigs were observed for clinical signs of diarrhoea from day 0-21. Ascoring system was applied to indicate its presence and severity. Thefollowing scoring system was used: 1=hard, 2=slightly soft, 3=soft,partially formed, 4=loose, semi-liquid and 5=watery, mucous-like.

Microbiology

A sample was serially diluted (1:10) in 9.0 ml aliquots of maximumrecovery diluent (MRD, Oxoid, Basingstoke, UK), and spread plated (0.1ml aliquots) onto selective agars. Lactobacillus spp. were isolated onde Man, Rogosa, Sharp agar (MRS, Oxoid) with overnight incubation at 37°C. in 5% CO2. The API 50 CHL (BioMerieux, France) kit was used toconfirm suspect Lactobacilli spp. E. coli species were isolated onMacConkey agar (Oxoid), following aerobic incubation at 37° C. for 18-24hours. Suspect colonies were confirmed with API 20E (BioMerieux,France).

Results Performance

Pigs fed LAM supplemented diets had an increased ADG (0.344 v 0.266,p<0.01) during days 7-14 and during the entire experimental period(0.324 v 0.232, p<0.01) compared to pigs offered diets with no LAM. Pigfed LAM supplementation had improved gain:feed ratio during days 7-14(0.763 vs. 0.569, p<0.001) and during the entire experimental period(0.703 v 0.646, p<0.05) compared to unsupplemented LAM diets. There wasa significant interaction (p<0.05) between LAM and FUC supplementationon ADG during days 14-21. Pigs offered the FUC diet had a significantlyhigher ADG than pigs offered the basal diet, however there was no effectof FUC when added to a LAM diet. There was no effect of LAM or FUCinclusion on average daily feed intake.

TABLE 19 The effect of seaweed extract on pig performance post weaning.Treatment Significance T1 T2 T3 T4 SEM LAM FUC LAM x FUC LAM − + − + FUC− − + + No of pens 12 12 12 12 Daily Gain (g/day) D 0-7 181 178 166 1850.025 ns ns ns D 7-14 268 320 265 368 0.022 ** ns ns D 14-21 418 459 475430 0.016 ns ns * D 0-21 288 319 302 328 0.012 * ns ns Food Intake(g/day) D 0-7 256 263 253 257 0.020 ns ns ns D 7-14 449 464 477 4570.027 ns ns ns D 14-21 604 686 673 619 0.024 ns ns ns D 0-21 436 471 467444 0.017 ns ns ns Gain to feed ratio (Kg/Kg) D 0-7 0.666 0.646 0.6460.679 0.055 ns ns ns D 7-14 0.579 0.707 0.561 0.818 0.049 *** ns ns D14-21 0.716 0.673 0.708 0.697 0.039 ns ns ns Days 0-21 0.654 0.675 0.6380.732 0.024 * ns ns Probability of significance; * = (P < 0.05), ** = (P< 0.01), *** = (P < 0.001).

Faecal pH, DM, Faecal Score

Pigs offered diets supplemented with LAM had an increased faecal DMcontent (28.64 v 26.24; p<0.05) compared to unsupplemented LAM diets.Pigs offered diets supplemented with LAM had a decreased faecal scoreduring days 7-14 (2.05 v 2.57; p<0.05). There was a significantinteraction between LAM and FUC inclusion on faecal score during theentire experimental period (days 0-21) (P<0.05). Pigs offered thecombination of LAM and FUC had a reduced faecal score compared to pigsoffered the FUC alone diet. However, there was no effect of LAMinclusion on faecal score compared to the basal diet.

TABLE 20 Effect of dietary treatment on faecal dry matter and faecalscore Treatment Significance T1 T2 T3 T4 SEM LAM FUC LAM x FUC LAM − +− + FUC − − + + No of pens 12 12 12 12 Faecal DM (g/Kg) 272.8 290.1252.2 282.8 10.2 * ns ns Faecal pH 6.42 6.25 6.19 6.31 0.109 ns ns nsFaecal score Days 0-7 2.45 2.61 2.49 2.07 0.149 ns ns ns Days 7-14 2.622.22 2.53 1.88 0.196 * ns ns Days 14-21 1.58 1.93 1.77 1.62 0.119 ns nsns Days 0-21 2.22 2.25 2.26 1.85 0.110 ns ns * Probability ofsignificance; * = (P < 0.05)

Microbiology and Volatile Fatty Acids (VFAs)

Pigs offered LAM diets had a reduced faecal E. coli population comparedto pigs offered diets with no LAM supplementation (7.22 vs. 7.84;p<0.05). There was a significant interaction (P<0.01) between LAM andFUC on faecal Lactobacilli populations. Pigs offered the FUC diet hadincreased Lactobacilli numbers compared to pigs offered the basal diet(9.22 v 8.93) however there was no effect of FUC on faecal lactobacillipopulations when included with LAM. There was no significant effect oftreatment on volatile fatty acid concentrations.

TABLE 21 The effect of dietary treatment on faecal Lactobacilli andEscherichia coli populations and faecal molar proportions of volatilefatty acids Treatment Significance T1 T2 T3 T4 s.e.m LAM FUC LAM x FUCLAM − + − + FUC − − + + E. coli 8.04 7.41 7.67 7.05 0.217 * ns nsLactobacilli 8.93 9.18 9.22 9.06 0.076 ns ns ** Total VFA (mmol/L) MolarProportions 141.4 134.5 130.1 110.4 9.197 ns ns ns Acetic Acid 0.5680.568 0.590 0.588 0.014 ns ns ns Propionic Acid 0.210 0.209 0.288 0.2190.007 ns ns ns Isobutyric acid 0.018 0.021 0.018 0.022 0.001 ns ns nsButyric Acid 0.144 0.135 0.152 0.123 0.011 ns ns ns Isovaleric Acid0.034 0.038 0.034 0.043 0.002 ns ns ns Valeric Acid 0.037 0.040 0.0380.038 0.003 ns ns ns Probability of significance; * = (p < 0.05), ** =(p < 0.01).

Pig offered LAM supplemented diets had improved Average Daily Gain (ADG)and gain to feed ratio (GFR) compared to pigs offered the unsupplementeddiets. This positive response to LAM may be due to the reduced E. colipopulation in the gut of these pigs. Diets supplemented with LAMresulted in pigs having a reduced faecal E. coli population whichresulted in reduced faecal DM and less diarrhoea (lower faecal score)during days 7-14, compared to pigs offered diets containing no LAM.Inclusion of LAM in the diet resulted in reduced Enterobacteriapopulation in the gut of the pig. Thus the improved performance seenwith pigs fed laminarin diets could be due to the associatedantimicrobial properties of LAM, which may result in an improved healthstatus and reduced coliform load in the gut of the pig. Modulation ofmucosal immunity by the binding of LAM to the specific receptors ofimmune cells may provide beneficial effects on pig health throughpreventing the colonization and proliferation of bacteria and thereforethe subsequent damage of the intestinal wall. The proliferation ofLactobacilli spp. in FUC supplemented diets would suggest that aproportion of the supplemented FUC is escaping hydrolysis in the foregutand passing into the colon for bacterial fermentation. Saccharolyticspecies of bacteria such as Lactobacilli spp. take part in the breakdownof complex carbohydrates. FUC is soluble in water making it a rapidlyfermentable carbohydrate source. Lactobacillus spp. have been reportedto ferment a number of monosaccharides which included L-fucose. In thecurrent study, it was found that the concentration of Lactobacillus spp.in the colon increased with the inclusion of FUC. Despite the increasein the Lactobacilli population, there was no dietary effect on VFAconcentration or profiles. The quantity of VFA produced in the largeintestine depends on the amount and composition of the substrate and onthe microflora present (MacFarlane and MacFarlane, 2003). However,faecal VFA concentrations may not be a totally accurate way todemonstrate fermentation intensity in the large intestine.

The combination diets of FUC+LAM were most effective at reducing postweaning diarrhoea. This could be attributed to a number of reasons.Firstly, it could be due to an immune response from feeding thecombination diets. Secondly, there was a numerical decrease in faecal E.coli numbers with the combination treatment. Pigs that express thesymptoms of diarrhoea harbour massive numbers of haemolytic E. coli.Therefore, a reduction in the numbers of E. coli present in the gutwould reduce the severity of diarrhoea and ultimately reduce pigletmorbidity post weaning.

Overall, the reduction in faecal E. coli population and the increase inADG and GFR suggest that LAM may provide a dietary means to improve guthealth post weaning. However, a combination of LAM and FUC is moreeffective at reducing diarrhoea.

Example 5 Experimental Design and Animal Diets

21 pigs with an initial weight of 17.9±2.2 Kg were assigned to one ofthe 3 dietary treatments: (T1) control; (T2) basal diet+300 ppm LAM;(T3) basal diet+600 ppm LAM. Experimental feeding continued for 21 daysad libitum. Diets were formulated to have similar digestible energy (DE)(14.4 MJ/Kg) and ileal digestible lysine (12.5 g/Kg).

Microbial and Volatile Fatty Acid (VFA) Analysis

Post-slaughter, digestive tract was removed by dissection and digestawas removed from the ileum. Each digesta sample was serially diluted inmaximum recovery diluent (MRD, Oxoid, Basingstoke, UK), and spreadplated onto selective agars. Bifidobacteria, Lactobacilli andEnterobacteria species were isolated according to the methods describedby Pierce et al. (2005). Digesta samples used to measure VFAconcentration were collected from the caecum and the same location inthe ileum and colon. VFA analysis was performed using gas liquidchromatography (GLC) according to the method described by Pierce et al.,(2005).

Collection of Tissue Samples and Tissue Challenge Procedure

Ileal and colonic tissues were sampled from the same location asdescribed for digesta samples. Excised tissues were emptied bydissecting them along the mesentery and rinsing them using sterilephosphate buffered saline (PBS) (Oxoid). Tissue sections 1 cm³, whichhad been stripped of the overlying smooth muscle were cut from eachtissue. Two sections from each tissue were placed in 1 ml of Dulbecco'sModified Eagle's Medium (DMEM) (Gibco), one in the presence of bacteriallipopolysaccharide (LPS) (Sigma Aldrich) at a concentration of 10 μg/ml.The other tissue sample was used as a control and incubated in sterileDMEM in the absence of LPS. Both challenged and unchallenged tissueswere incubated at 37° C. for 90 minutes before being removed, blotteddry and weighed. Approximately 1-2 g of porcine ileum and colon tissueswere cut into small pieces and collected in 15 ml of RNAlater® (AppliedBiosystems, Foster City, Calif.). RNAlater® was removed before storingthe samples at −80.0 until used for RNA extraction.

Preparation of Unchallenged Tissue for Quantitative Real Time PCR(qRT-PCR)

Ileal and colonic tissues was stabilised in RNAlater® solution andstored overnight at 4° C. The following day, RNAlater® was removed andsamples were stored at −86° C. prior to RNA extraction.

RNA Extraction and cDNA Synthesis

Tissue samples for RNA extraction were removed from −86° C. andhomogenised. 500 μl of lysis solution/2-ME was added to each sample andthese were mechanically disrupted using one 5 mm stainless steel beadper sample. These were then placed in a Tissue Lyser (Qiagen) andlysates were homogenised for 3 mins and transferred to a GenEluteFiltration Column (Sigma Aldrich) and RNA was extracted. 1 □g of totalRNA was used for cDNA synthesis using oligo(dT)₂₀ primer in a finalreaction volume of 20 μl with Superscript™ III First-Strand synthesissystem for reverse transcriptase-polymerase chain reaction (RT-PCR)(Invitrogen Life Technologies, Carlsbad, Calif.). At the last step ofcDNA synthesis, treatment with E. coli RNase H (Invitrogen Corp.) wasperformed to digest the remaining RNA/mRNA template, resulting in theproduction of the single-stranded cDNA template for subsequent qRT-PCRreactions.

Quantitative Real-Time PCR (qPCR) and Normalization of qPCR Data

All porcine primers for the cytokine genes interferon gamma (IFN-γ),interleukin-1α (IL-1α), IL-6, IL-8, IL-10, IL-17, tumour necrosis factor(TNF-α), the mucin genes (MUCs 1, 2, 4, SAC, 12, 13 and 20) and threereference genes, β-actin (ACTB), Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and Peptidylprolyl isomerase A (PPIA), weredesigned using Primer Express™ (PE Applied Biosystems, Foster City,Calif.) and synthesised by MWG Biotech (Milton Keynes, UK). Thesereference genes were previously validated for use in porcine tissue andqPCR was then carried out on the cDNA using the ABI PRISM 7900HT Fastsequence detection system for 96-well plates (Applied Biosystems, FosterCity, Calif.). All samples were prepared in duplicate using SYBR GreenFast PCR Master Mix (Applied Biosystems, Foster City, Calif.), cDNA astemplate and specific primers for the genes selected. For each reaction5 μl cDNA, 1.2 μl (forward and reverse primer mix, 5 μM), 10 μl FastSYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, Calif.)was added and made up to a final volume of 20 μl. The two step PCRprogram was as follows: 95° C. for 10 minutes for 1 cycle followed by95° C. for 15 seconds and 60° C. for 1 minute for 40 cycles. The raw Ctvalues for the reference genes were converted to relative quantitiesusing the formula Q=E^(Δ)Ct where E is the PCR efficiency of the assayand ^(Δ)Ct is the value calculated for the difference between the lowestCt value for each gene minus the Ct value of the sample in question. Therelative quantities of the endogenous controls were then analysed forstability in geNorm (Vandesompele et al., 2002). The stability ‘M’ valuegenerated by the geNorm application for the selected endogenous controls(ACTB, GAPDH and PPIA) indicated their suitability as endogenouscontrols for these intestinal samples. The geometric mean of therelative quantities for ACTB, GAPDH and PP/A (normalisation factor) wasthen calculated using geNorm. The relative quantities of each targetgene were divided by the normalisation factor (obtained in geNorm) forthat sample to give the final normalised relative expression.

Results

In this study, LAM from Laminaria digitata did not affect performance,nutrient digestibility or selected bacteria in the ileum, but it diddecrease Enterobacteriaceae in the colon. Of particular interest was theimpact of LAM on cytokine gene expression in the ileum and colonfollowing in-vitro challenge with lipopolysaccharide (LPS).

Animal Performance and Nutrient Digestibility

There was no effect on performance (food intake, daily gain or foodconversion ratio) or nutrient digestibility coefficients (DM, OM, ash, Nor GE) with increasing LAM.

Microbiology and Volatile Fatty Acids (VFAs)

Increasing the level of LAM from 0-600 ppm had no effect on theBifidobacteria, Lactobacilli or Enterobacteriaceae Populations in theIleum (p>0.05). There was a Decrease in Enterobacteriaceae populationswith increasing LAM. The potential to reduce harmful Enterobacteriaceaestrains, without influencing Bifidobacteria and Lactobacilli numbers isof great significance as pathogenic bacteria increase mortality rates.In context, results indicate that the optimum LAM inclusion rate is 300ppm.

TABLE 22 The effect of increasing LAM on selected microbial populationsin the ileum, proximal and distal colon and total VFAs in the ileum,caecum and proximal colon of the pig. Significance LAM 0 ppm 300 ppm 600ppm SEM Linear Quadratic (Log₁₀ CFU/g) Ileum Bifidobacteria 4.94 5.445.51 0.708 ns ns Lactobacilli 4.14 5.15 5.40 0.565 ns nsEnterobacteriacae 2.24 3.35 2.86 0.839 ns ns Colon Bifidobacteria 7.377.29 7.46 0.365 ns ns Lactobacilli 7.89 8.16 8.19 0.221 ns nsEnterobacteriacae 5.42 3.87 4.24 0.358 * * Total VFAs Ileum 10.47 14.8414.26 2.60 ns ns Caecum 173.5 189.2 194.4 9.70 * ns Colon 185.4 146.4161.8 13.79 ns ns

Probability of significance: * P<0.05, ** P<0.01, *** P<0.001,ns=non-significant P>0.05 There were no significant effects ofincreasing dietary inclusion levels of LAM on total VFAs, in the ileumor colon. There was a significant increase in total VFAs with increasinglevels of LAM in the caecum (p<0.05), the main site of VFA production.There was no significant alteration in digesta pH recorded from any theintestinal region.

Cytokine Gene Expression

There were no effects of LAM in unchallenged ileum or colon tissue forany of the cytokines analysed. This overall lack of an effect on theseinflammatory markers implies that the presence of LAM in the diet didnot elicit any negative effects. To mimic the response of the ileal andcolonic tissues of animals exposed to LAM to a microbial challenge,these tissues were subsequently incubated with LPS ex-vivo. While noeffect was observed in the ileum, a significant challenge effect wasobserved for IL-6 and IL-8 gene expression in the colon ofLPS-challenged tissue. LAM inclusion levels at 300 ppm lead to anincrease in IL-6 expression (p<0.05), whilst a linear increase in IL-8gene expression was observed (p<0.05). These data suggest that dietaryLAM could enhance the pro-inflammatory response to microbial challenge.The potential benefit of this enhanced gene up-regulation of IL-6 andIL-8 cytokines following the LPS challenge are significant for the hostas IL-6 is a pro-inflammatory cytokine that plays an important role inacute inflammation in the early immune response. Similarly the chemokineIL-8 also plays an important role in inflammation and is responsible forneutrophil recruitment and activation to the initial site of infection.While exposure to LAM alone did not stimulate pro-inflammatory cytokineproduction in the gastric mucosa, it enhanced the LPS inducedpro-inflammatory cytokine production.

TABLE 23 The effect of increasing LAM from Laminaria digitata on theimmune response in unchallenged ileum and colon tissues. SignificanceLAM 0 ppm 300 ppm 600 ppm SEM Linear Quadratic Ileum IFN-γ 1.000 1.2780.841 0.233 ns ns IL-1α 1.000 1.141 0.597 0.148 ns ns IL-6 1.000 1.6810.908 0.252 ns ns IL-8 1.000 1.003 0.475 0.245 ns ns IL-10 1.000 1.1280.646 0.229 ns ns TNF- α 1.000 1.023 0.805 0.133 ns ns Colon IFN-γ 1.0001.217 1.148 0.286 ns ns IL-1α 1.000 0.716 0.851 0.144 ns ns IL-6 1.0001.579 1.788 0.434 ns ns IL-8 1.000 1.245 1.137 0.224 ns ns IL-10 1.0001.029 0.843 0.285 ns ns TNF-α 1.000 1.400 1.446 0.301 ns ns Probabilityof significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns =non-significant p > 0.05

TABLE 24 The effect of LAM from Laminaria digitata on immune response inthe ileum and colon following an ex-vivo LPS tissue challenge.Significance LAM 0 ppm 300 ppm 600 ppm SEM Linear Quadratic Ileum IFN-γ1.000 0.927 1.098 0.223 ns ns IL-1α 1.000 1.072 1.039 0.161 ns ns IL-61.000 1.352 1.143 0.266 ns ns IL-8 1.000 1.186 0.903 0.362 ns ns IL-101.000 1.198 1.076 0.160 ns ns TNF- α 1.000 0.819 0.921 0.108 ns ns ColonIFN-γ 1.000 2.051 1.614 0.385 ns ns IL-1α 1.000 0.983 1.242 0.199 ns nsIL-6 1.000 1.846 0.830 0.272 * * IL-8 1.000 1.590 1.948 0.303 * — IL-101.000 0.936 1.039 0.256 ns ns TNF- α 1.000 1.557 0.938 0.250 ns nsProbability of significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns= non-significant p > 0.05

Mucin Gene Expression

Dietary factors such as fibre, protein and anti-nutritional factors areknown to directly influence the synthesis and secretion of mucin fromgoblet cells and the recovery of mucin in digesta (Montagne et al.,2004). All 7 mucin gene transcripts were reliably detected in theporcine colon but only five of the seven were accurately quantifiable inthe ileum. An increase in MUC2 was observed in the ileum of pigssupplemented with LAM at 300 ppm (p=0.05) relative to the controlanimals. This increased MUC2 expression was not observed at the higherdietary inclusion level (600 ppm). LAM supplementation had no effect onthe remaining detectable mucins (MUC4, MUC12, MUC13 and MUC20) in theileum. In the colon, dietary supplementation with LAM at an inclusionlevel of 600 ppm, significantly increased MUC2 (quadratic; P<0.05) andMUC4 (quadratic; P<0.05) expression but had no effect on the expressionof any of the remaining mucin genes at this site. Diets containingbeta-glucans also affect the quality and quantity of mucin production ofthe jejunum, ileum, caecum and colon in the murine model (Deville etal., 2007).

TABLE 25 Effect of LAM from Laminaria digitata on mucin gene expressionin ileum and colon. Significance LAM 0 ppm 300 ppm 600 ppm LinearQuadratic Ileum MUC2 1.000 0.000 * * MUC4 1.000 0.000 ns ns MUC12 1.0000.000 ns ns MUC13 1.000 0.000 ns ns MUC20 1.000 0.000 ns ns Colon MUC11.000 0.902 1.270 ns ns MUC2 1.000 0.726 1.207 * * MUC4 1.000 0.9811.351 * * MUC5AC 1.000 2.134 0.285 ns ns MUC12 1.000 0.957 1.120 ns nsMUC13 1.000 0.924 1.077 ns ns MUC20 1.000 1.107 1.063 ns ns Probabilityof significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns =non-significant p > 0.05

Optimum Inclusion Level

300 ppm LAM is sufficient to ‘prime’ the immune system in an ex-vivoLPS-challenge.

Example 6

Immune capacity can be modulated by nutritional interventions with LAMand/or FUC leading to a reduction in Porcine circovirus type 2 (PCV2)viral load in experimentally infected snatch farrowed pigs andameliorating the effects of post-weaning multisystemic wasting syndrome(PMWS) in pigs.

Results Immunofluorescent Detection of PCV2 Antigen in Tissues

PCV2 antigen was detected in tissue sections from necropsied animals(liver, lung, kidney, spleen, ILN, MLN) by immunofluorescence usingPCV2-specific monoclonal antibody.

-   -   In the basal diet, 5 of 6 animals were euthanized.    -   In the basal diet+LAM and FUC treatment, one of six animals was        euthanised.    -   In the basal diet with LAM and FUC and WPI, one of the six pigs        was euthanised during of the experiment. Tissues from this        animal contained high levels of PCV2 antigen. The remaining five        animals appeared healthy at the end of the experiment. They had        seroconverted and gained weight.

Determination of PCV2-Specific Antibody Titre

Referring to FIG. 1, there is shown the PCV2-specific antibody titre ofsera, which was determined by IPMA.

Intra Group Analysis

-   -   Basal diet: 5 of 6 piglets gave a poor PCV2 antibody response        and all had PCV2 antigen indicative of disease in analysed        tissue sections. The remaining animal (Tag 10) seroconverted to        a reasonable PCV2-specific antibody titre. This animal remained        healthy throughout the duration of the experiment.    -   Basal diet+LAM and FUC treatment: 1 of 6 pigs had to be        euthanised before the end of the experiment (Tag 30). This        animal had the lowest PCV2-specific antibody titre of all        animals in this group and PCV2 antigen levels in tissues were        indicative of PCV2 associated disease. However, the antibody        titre of this animal was higher than that of piglets that        developed disease in Group 1.    -   Basal diet+with LAM and FUC+WPI: One of the six pigs was        euthanized before the end of the experiment (Tag 26). All of the        remaining five animals were healthy at the end of the        experiment, had seroconverted and gained weight. Two animals        (Tag 22 and 25) had high levels of PCV2 antigen in its tissues.

Inter Group Analysis

At 21 and 28 days post-infection (PI), the mean PCV2-specific antibodytitre of animals in treatment 2 (+LAM and FUC; See FIG. 2) and 4 (+LAMand FUC+WPI, See FIG. 3) were significantly higher (p<0.05) than animalsfed the basal diet and piglets fed the basal+WPI. These results suggestthat LAM and FUC supplementation of pig feed, alone or in conjunctionwith WPI, boosts the humoral response of PCV2 infected pigs.

Lymphocyte Numbers

Referring to FIG. 4, it can be seen that by day 28 post-infection (PI),the basal diet feed group had significantly lower percentage lymphocytecell population than the three supplemented diets. Neither supplementwas significantly different from each other. At 14 days PI, piglets feda diet supplemented with LAM+FUC had significantly greater (p<0.05)percent eosinophils than all the other groups, as illustrated in FIG. 5.After this time, no significant difference was detected.

Analysis of Animal Weights

The weights of pigs were recorded weekly. With reference to FIG. 6, itcan be seen that the average terminal weights of piglets in groups 2(+LAM and FUC), 3 (+whey protein isolate) and 4 (+LAM and FUC+wheyprotein) were greater than those fed the basal diet. Animals in groups 2and 4 were significantly heavier than piglets fed the basal diet.

Analysis of Animal Body Temperatures

Body temperatures were also monitored throughout the study. At 17 daysPI, animals in group 3 (+whey protein isolate) and group 4 (+LAM and FUCand whey protein isolate) had significantly lower mean temperatures(38.33 and 38.02° C., respectively) than piglets fed the basal diet(39.82° C., p<0.05). No significant difference in mean body temperatureswere observed between the individual feed groups at 24 days PI.

Analysis of Viral Shedding

Quantitative PCR was performed on all faeces samples to estimate viralload and shedding. As illustrated in FIG. 7, by day 10 PI, the averagePCV2 DNA copy number detected in animals in Group 3 (+whey proteinisolate) was significantly lower (p<0.05) than the 3 other feed groups.On 35 days 20 and 24 PI, piglets fed the LAM and FUC supplemented diethad significantly lower PCV2 DNA copies than piglets in Groups 1 and 3.Animals fed LAM and FUC and whey protein isolate contained significantlylower PCV2 DNA copy numbers than those in the basal diet (p<0.05). Byday 27 PI, all supplemented diets had significantly lower copies of PCV2DNA (p<0.05) than those on the basal diet. The lowest average PCV2 DNAcopy number was detected in the faeces samples of piglets fed LAM+FUC,as seen in FIG. 8. These results indicate that supplementing pig feedwith LAM+FUC, WPI, or both in combination led to a significant reductionin viral shedding of PCV2 under these experimental conditions.

TABLE 26 Immunofluorescence detection of PCV2 antigen in tissuesections. Tag Group Disease PM Number No No Liver Lung Kidney Spleen ILNMLN status Basal diet 07-11887 3 1 1+ 3+ 2+ 3+ 3+ 2-3+ Y 07-11872 5 1 3+4+ 2-3 4+ 1+ 2+ Y 07-11893 7 1 2-3+ 4+ 2-3+ 3+ 4+ 4+ Y 07-11894 10 1 2+3-4+ 1-2+ 3+ No tissue 2-3+ Y 07-11881 20 1 1+ 1+ 1-2+ 1+ 2+ 2-3+ N07-11889 35 1 2+ 2-3+ 1-2+ 1-2+ 3+ 3+ Y Basal diet + 1 g/Kg LAM and FUC07-11888 2 2 +/− +/− 1+ 1+ 2+ 1-2+ N 07-11886 8 2 −ve −ve +/− 1-2+ 1+1-2+ N 07-11871 14 2 +/− +/− 1+ 1+ 1-2+ 1+ N 07-11878 24 2 −ve −ve +/−1+ 1+ 1-2+ N 07-11890 30 2 2-3+ 3+ 2+ 2-3+ 4+ 3+ Y 07-11882 31 2 +/− +/−1+ 1+ 1-2+ 2+ N Basal diet + 80 g/Kg whey protein isolate 07-11869 9 31+ 1-2+ 1+ 2+ 3+ 3-4+ Y 07-11884 15 3 2-3+ 3-4+ 3+ 3+ 3-4+ 3-4+ Y07-11863 19 3 1+ 1+ 2+ 1+ 2+ 3+ Y 07-11892 27 3 2-3+ 4+ 2+ 2-3+ 4+ 3+ Y07-11859 29 3 −ve +/− 2+ 2+ 3+ 3-4+ Y 07-11858 38 3 −ve −ve −ve +-1+ 1+1+ N Basal diet + 1 g/Kg LAM and FUC + 80 g/Kg whey protein isolate07-11861 1 4 +/− + 1+ + 2-3+ 1-2+ N 07-11870 4 4 −ve −ve 1-2+ 1+ 2+ 2+ N07-11867 6 4 −ve −ve 1-2+ +/− 1-2+ 2+ N 07-11862 22 4 +/− + 1+ 1-2+ 2-3+3+ Y 07-11860 25 4 2+ 3+ 3+ 2-3+ 4+ 4+ Y 07-11895 26 4 2-3+ 3-4+ 2+ 3+No tissue 2+ Y Scores ≧3+ = high level of viral antigen indicative ofPCV2-associated disease.

Cytokine PCR Results

Cytokine mRNA for IL-2, TNF-α and IL-4 were quantified using anestablished two-step reverse transcription PCR (rtPCR) assay. Resultsindicated that there were no significant differences in the profile ofthese cytokines between the different feed treatments.

Quantification of PCV2 DNA in Tissue Homogenate

Tissue homogenate pools (10% w/v) were prepared from the liver, lung,spleen, kidney, mesenteric and inguinal lymph nodes of each animal. PCV2DNA was quantified using an established quantification PCR method(qPCR). The highest amounts of PCV2 DNA were detected in animals thatreceived the basal diet (Group 1). Pigs fed the diet supplemented withWPI (Group 3) also had higher quantities of PCV2 DNA than groups thatreceived the basal diet supplemented with either LAM+FUC (Group 2) orLAM+FUC in combination with WPI (Group 4). These results comparefavourably with the levels of the immunofluorescence-based detection ofPCV2 antigen in the animal tissues. The least amount of PCV2 antigen wasdetected in animals fed a diet supplemented with LAM+FUC alone or inconjunction with WPI compared to the other two groups (i.e.basal/basal+WPI).

This invention reduces Porcine circovirus type 2 (PCV2) viral load inexperimentally infected snatch farrowed pigs and ameliorates the effectsof post-weaning multisystemic wasting syndrome (PMWS) in pigs.

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We claim:
 1. A method for improving or maintaining the health,structure, function, immunity or performance of the progeny of amaternal animal or human; or preventing, treating or amelioratingdisorders in structure or function or immunity in the progeny of amaternal animal or human, to levels equivalent to or greater than thoseachieved by direct administration to each individual progeny at weaningor later; the method comprising administering a composition comprisingat least one glucan, at least one fucan, or at least one glucan and atleast one fucan to the maternal animal or human.
 2. The method accordingto claim 1, wherein the at least one glucan is a beta glucan.
 3. Themethod according to claim 1, wherein the at least one glucan is beta(1→3, 1→4) glucan or beta (1→3, 1→6) glucan.
 4. The method according toclaim 1, wherein the at least one glucan is laminarin.
 5. The methodaccording to claim 1, wherein the at least one fucan is an alpha-fucan.6. The method according to claim 1, wherein the at least one glucanand/or the at least one fucan is isolated from a brown macroalga of theclass Phaeophycea.
 7. The method according to claim 1, wherein the atleast one glucan and/or the at least one fucan is isolated from a redalga selected from Florideophyceae.
 8. The method according to claim 1,wherein the composition is administered to the maternal animal or humanperinatally and/or prenatally, and/or postnatally.
 9. The methodaccording to claim 1, wherein the composition is administered daily tothe maternal animal or human.
 10. The method according to claim 1,wherein the composition is administered to the maternal animal or humanin an amount such that about 2-40 milligrams of fucan per kilogram ofbody weight is administered to the maternal animal or human.
 11. Themethod according to claim 1, wherein the maternal animal or human is amonogastric animal selected from the group consisting of pigs, poultry,fish, cats, dogs and humans; and/or the animal is a hind-gut fermenterselected from the group consisting of horses and rabbits; and/or theanimal is a foregut fermenter selected from the group consisting ofsheep.
 12. The method according to claim 1, wherein the disorder ofstructure or function is prevented, treated or ameliorated by alteringimmunological function and/or the immunological profile; and/or alteringimmune gene expression and/or the expression or secretion of signallingproteins; and/or altering the expression of intra-cellular and/orextra-cellular receptors.
 13. The method according to claim 12, whereinimmunological function and/or immunological profile is improved byaltering leukocyte numbers and/or leukocyte function and/or leukocytephenotype and/or leukocyte trafficking and/or leukocyte distributionand/or cytokine gene expression and/or cytokine secretion and/orcytokine receptor expression.
 14. The method according to claim 13,wherein leukocyte numbers are reduced and/or phagocytic activity isincreased.
 15. The method according to claim 13, wherein the cytokine isselected from pro-inflammatory and/or anti-inflammatory cytokines. 16.The method according to claim 1, wherein the disorder of structure orfunction in the progeny is a chronic disease selected from the groupconsisting of Crohn's disease, Irritable Bowel syndrome, and Colitis.17. The method according to claim 1, wherein the disorder of structureor function in the progeny is selected from the group consisting of anautoimmune, inflammatory, autoinflammatory, and atopic disease.
 18. Themethod according to claim 1, wherein the disorder of structure orfunction in the progeny animal is prevented, treated or ameliorated byincreasing the concentration of immunoglobulin in the colostrum or milkof a maternal animal or human.
 19. The method according to claim 1,wherein the disorder of structure or function in the animal progenyanimal is prevented, treated or ameliorated by decreasing bacterialinfection and/or decreasing viral infection.
 20. The method according toclaim 1, wherein the disorder of structure or function in the progeny isprevented, treated or ameliorated by increasing the expression ofmucins.
 21. The method according to claim 1, wherein the disorder ofstructure or function in the progeny is one which gives rise to wastingaway of muscle and/or fat tissue.
 22. The method according to claim 1,wherein the disorder of structure or function in the progeny isprevented, treated or ameliorated by decreasing infection or load ofviral pathogen.
 23. The method according to claim 24, wherein the viralpathogen is a non-enveloped virus.
 24. The method according to claim 1,wherein the disorder of structure or function in the progeny isprevented, treated or ameliorated by increasing the production ofstraight-chain volatile fatty acids and/or reduction of branched-chainfatty acids.
 25. The method according to claim 1, wherein the at leastone fucan is fucoidan.
 26. The method according to claim 1, wherein theat least one glucan and/or the at least one fucan is derived from atleast one family selected from the group consisting of Laminariaceae,Fucaceae and Lessoniaceae.
 27. The method according to claim 1, whereinthe at least one glucan and/or the at least one fucan is selected fromat least one species from the group consisting of Ascophyllum species;Laminaria species and Sargassum species.
 28. The method according toclaim 1, wherein the at least one glucan is derived from a species offungi.
 29. The method according to claim 1, wherein the at least oneglucan is derived from yeast.
 30. The method according to claim 1,wherein the at least one glucan is derived from Saccharomycescerevisiae.
 31. The method according to claim 1, wherein the disorder ofstructure or function in the progeny is selected from the groupconsisting of psoriasis, rheumatoid arthritis, psoriatic arthritis,atopic dermatitis and juvenile idiopathic arthritis.
 32. The methodaccording to claim 1, wherein the disorder of structure or function inthe animal progeny is prevented, treated or ameliorated by decreasinginfection selected from the group consisting of Escherichia coliinfection, Campylobacter infection, Salmonella, porcine circovirusinfection and PCV-2 infection.
 33. The method according to claim 1,wherein glucans are derived by approaches which include syntheticchemistry and biotechnology-related approaches.
 34. The method accordingto claim 1, wherein fucans are derived by approaches which includesynthetic chemistry and biotechnology-related approaches.
 35. The methodaccording to claim 1, wherein administration of the composition to thematernal animal or human confers benefits to a single progeny from thematernal animal or human.
 36. The method according to claim 1, whereinadministration of the composition to the maternal animal or humanconfers benefits to multiple birth progeny offspring from the maternalanimal or human.
 37. The method according to claim 1, whereinadministration of the composition to the maternal animal or humanconfers benefits to multiple birth progeny offspring such that 2 or moreprogeny offspring from the maternal animal or human are affected. 38.The method according to claim 1, wherein the disorder of structure orfunction gives rise to early life mortality in progeny.
 39. The methodaccording to claim 1, wherein early life mortality is reduced duringperinatal, prenatal and postnatal periods, during gestation, pregnancy,viparity, ovoviviparity or viviparity, surrounding the time of birth,hatching or spawning or during post birth, post hatch, neonatal orweaning periods, optionally with reductions in morbidity, mortality,numbers of stillborn, un-hatched, un-fertilized or mummified offspringor reductions in pre- or post-birth or hatch deaths.
 40. The methodaccording to claim 1, wherein parity is improved.
 41. The methodaccording to claim 1, wherein performance is increase throughout theanimals lifetime, during the post birth period, weanling period,juvenile period, fattening period, finisher period, slaughter ageperiods, adult stage and periods during which animals grow to reachtheir genetic growth potential.
 42. The method according to claim 1,wherein immunoglobulins are increased in blood serum of the progenyoffspring of the maternal animal.
 43. The method according to claim 1,wherein the uniformity of progeny offspring weaning weight issignificantly improved.
 44. The method according to claim 1, wherein thepercentage of offspring weaned per maternal animal on weaning day isincreased.